Bacteria of the genera Rhizobium and Bradyrhizobium possess the ability to infect plants and establish a nitrogen-fixing symbiosis. This process is termed nodulation and the morphological structure formed on the root in which the bacteria reside is termed a nodule. The formation of a nodule is a developmental process both from the standpoint of the bacteria and the plant. Each step in the process likely involves one or more bacterial and plant genes.
The genes in rhizobia (i.e., bacteria of the genera Rhizobium and Bradyrhizobium) involved in nodule formation and function are sometimes referred to as sym (for symbiotic) genes. The sym genes are further classified into three broad categories: nif, fix, and nod genes. The distinction between these genes is not always clear. For the purposes of this invention, nif genes include those that are responsible for production of the nitrogen-fixing enzyme, nitrogenase (i.e., nif KDH) and all other genes that are analogous to nif genes already identified in Klebsiella pneumoniae. The fix genes are those genes necessary for nitrogen fixation but which are not comparable to the nif genes of K. pneumoniae. The nod genes are those genes involved in the formation of the nodule.
The ability of a specific Rhizobium or Bradyrhizobium to infect a leguminous host is generally restricted; only particular Rhizobium/Bradyrhizobium species-host species combinations are possible. This specificity of interaction is controlled by both plant and bacterial encoded genes. In general, Bradyrhizobium can nodulate a wider range of hosts than can be modulated by Rhizobium species.
Knowledge of the nodulation genetics of Rhizobium species is relatively well advanced, but the same cannot be said with regard to the taxonomically distinct Bradyrhizobium species. Both groups of bacteria contain one set of genes (nodABCDIJ) that are required for nodulation of all hosts; the so-called "common nodulation genes" due to their ability to functionally complement nodulation defective mutants in other rhizobia species (Long, et al. 1982, Nature 298:485; Banfalvi, et al., 1983, Mol. Gen. Genet. 203:42; Downie, et al., 1983, EMBO J. 2:974; Schofield, et al., 1984, Plant Mol. Biol. 3:3; Russell, et al., 1985, J. Bacteriol. 164:1301; Lamb, et al., 1986, Mol. Gen. Genet. 202:512; Noti, et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:7379; Marvel, et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:5841; Scott, K. R., 1986, Nucl. Acids Res. 14:2905.) A second set of genes has been identified in Rhizobium species that are necessary for nodulation of a particular host. These genes have been termed host specificity (hsn) genes. For example, hsnABCD=nodEFGH (Horvath, et al., 1986, Cell 46:335; Kondorosi, et al., 1984, Molec. Gen. Genet. 193:445.)
Rhizobium sp. strain MPIK3030, also referred to as NGR234, is capable of nodulating a wide range of leguminous hosts (Trinick, M. J., 1980, J. Appl. Bacteriol. 49:39). The ability of this bacteria to nodulate many hosts is apparently due to the presence of several hsn gene loci (Broughton, et al., 1986, J. Cell. Biol. 102:1173.) One such locus, affecting the ability to nodulate siratro (Macroptilium atropurpureum) has been cloned from this organism (Bachem, et al., 1986, Mol. Gen. Genet. 203:42; Bassam, et al., 1986, Mol. Gen. Genet. 203:49.) The foregoing papers by Trinick, Broughton, Bachem and Bassam et al. are each hereby incorporated herein by reference.
The foregoing paper by Russell et al. describes recombinant DNA clones, including a clone pRjUT10, comprising a series of contiguous HindIII fragments having sizes of 3.3, 5.6, 3.9, 1.7, 2.3, 4.5, 4.6 and 4.3 kilobases (kb). The 5.6, 3.9 and 1.7 kb fragments were designated a nod region, but no special significance was attributed to the adjoining 3.3 kb fragment.
According to the present invention, it has been found that cloned siratro hsn genes from Rhizobium sp. MPIK 3030 will hybridize to genomic DNA from B. japonicum. B. japonicum will nodulate siratro in addition to its preferred host, soybean. This invention relates to the identification and isolation of this broad host range hsn locus from B. japonicum. This gene is wholly contained in the 3.3 HindIII fragment obtained by Russell et al.